PROJECT 4: PROJECT SUMMARY Dramatic progress has been made in recent years in treating patients with advanced melanoma, particularly with inhibitors of PD-1 or PD-L1, resulting in prolonged survival and a sharp rise in the number of patients living with metastatic melanoma. However, over half the patients do not respond to PD-1/-L1 inhibitors, and tumor regrowth is seen in about half the patients by two years. Thus, there is great need to understand mechanisms of resistance and to develop new approaches to overcome resistance. A well-documented mechanism of resistance to PD-1/L1 inhibitors is paucity of tumor infiltrating T cells (TILs). We have found that targeting tumor associated macrophages by inhibition of the colony stimulating factor-1 receptor (CSF1R) results in tumor regression in a T cell independent fashion and induces increased TNF?, IL-1?, IFN? expression and neutrophil recruitment. Similarly, antibodies that activate CD40 also result in macrophage modulation and tumor regression and synergize with CSF1R. When used together in mice, these treatments stimulate a T cell-dependent anti- tumor response. We hypothesize that triple therapy (inhibitors of CSF1R and PD1 with CD40 agonists) may be even more effective at eliciting anti-tumor responses to tumors that were at one time poorly infiltrated and/or recognized by T cells. We will test the key hypothesis that stimulating both innate and adaptive immunity leads to more robust anti-tumor immunity than those that preferentially target T cells or myeloid cells alone. We will utilize a series of novel immune competent murine models of resistance to PD-1/PD-L1 inhibitors generated at Yale with driver mutations that mimic human melanomas (e.g., BrafV600E, NrasQ61R, loss of Cdkn2a, Pten, p53, and gain of ?-catenin). We will study the effects of CSF1R inhibitors and CD40 agonists with and without PD-1 inhibitors to determine activity and toxicity of doublet and triplet regimens in murine models with various genetic backgrounds (Aim 1). In Aim 2 we will study the mechanisms of sensitivity or resistance to the various combinations to facilitate predictive biomarker studies for humans treated with these regimens. We will conduct a phase I/IB clinical trial (Aim 3) of a CSF1R mAb in combination with a CD40 agonist with concurrent dose escalation of a triplet regimen that will include the addition of nivolumab. Expansion of the optimal regimen will be conducted using a Simon two stage design. Patients eligible for the trial will have advanced melanoma that has progressed on prior PD-1/PD-L1 based regimens. Tumors from patients treated on this trial will be characterized for tumor associated macrophage subsets and TIL subsets using state-of-the-art technologies. The collaborative team, availability of clinically-relevant animal models and access to patients and their tumors will thus enable us to go back and forth between the clinic and the lab, facilitating personalized approaches for patients with tumors poorly responsive to PD-1/PD-L1 inhibitors. Results from these studies will be helpful for overcoming resistance to PD-1/PD-L1 inhibitors in other tumor types as well.